Our objective was to characterize changes in vitamin D metabolites of plasma in Jersey cows fed a prepartum low-calcium diet. Eight cows were fed a high-calcium diet (80 g/day) and eight were fed a low-calcium diet (8 g/day) at least 14 days before parturition. Calcium concentrations in plasma decreased after initiation of feeding either diet, but cows fed low-calcium diet tended to have lower prepartum calcium and phosphorus and greater peripartal calcium in plasma. Hydroxyproline in plasma was greater during peripartal period in cows fed low-calcium diet. Prepartum 1,25-dihydroxyvitamin D in plasma tended to be greater in cows fed low calcium. Increases in 1,25-dihydroxyvitamin D were only 2 and 3 days after initiation of the low-calcium diet; during the first 2 days after parturition, however, 1,25-dihydroxyvitamin D tended to be lower in those cows fed low calcium. As parturition neared, 24,25-dihydroxyvitamin D tended to be lower in cows fed the low calcium-diet. Usual early postpartum changes in calcium phosphorus, magnesium, 1,25-dihydroxyvitamin D, and hydroxyproline were seen during first few days after initiation of feeding low calcium. Thus, we propose that the preventative action of the low-calcium diet is associated with preparation of the calcium homeostatic mechanism several days before the calcium demand of initiation of lactation.